Currently, the intensive chemotherapy remains the first line treatment for B cell acute lymphoblastic leukemia (B-ALL). Although these regimens have significantly improved patient outcomes, their use is associated with debilitating morbidities and fatal relapses, highlighting the great need in new agents that target essential survival signals in leukemia. Thus, the overall goal of my project was to gain insights into the signaling abnormalities that regulate aberrant proliferation and survival of B-ALL cells in an effort to identify novel targets in this malignancy. This study demonstrated that pre-B cell receptor (pre-BCR)-independent spleen tyrosine kinase (SYK) activity was required for the survival and proliferation of a p53-/-PrkdcSCID/SCID mouse model of B-ALL. I extended this discovery to human disease, demonstrating that SYK was activated in primary B-ALL, independent of the pre-BCR expression. The small molecule SYK inhibitor fostamatinib (fosta) significantly attenuated proliferation of 79 primary diagnostic B-ALL samples at clinically achievable concentrations. Importantly, fosta treatment reduced dissemination of engrafting B-ALL cells into the spleen, liver, kidney and central nervous system (CNS) in a NOD.Prkdcscid/scidIl2rgtm1Wjl/SzJ xenotransplant model of B-ALL. Analysis of signaling abnormalities using a high-throughput phospho-flow cytometry platform demonstrated that pediatric and adult B-ALL samples exhibit variable basal activation of BCR, iii PI3K/AKT/mTOR, MAPK and JAK/STAT pathways. Importantly, we identified that fosta-mediated inhibition of SYK, PLC2, CRKL and EIF4E phosphorylation in B-ALL was predictive of its anti-leukemic activity, and was distinct from the cellular actions of other small molecule inhibitors of key nodal signaling pathways. Examination of molecular mechanism of fosta action by gene expression profiling revealed transcriptional effects of fosta treatment that included, most notably, potent inhibition of pathways involved in lymphocyte activation and inflammation. In conclusion, this study demonstrates that SYK signaling is crucial for B-ALL survival and provides detailed characterization of cellular and molecular mechanisms of fosta action in B-ALL. These data argue in favor of testing small molecule SYK inhibitors in pediatric and adult B-ALL.

An 11 year old, hispanic girl with a history of B-cell acute lymphoblastic leukemia was admitted to the hospital for symptoms compatible with Bartonella henselae infection. The first molecularly diagnosed case of disseminated Bartonella henselae infection was reported in an immunocompromised patient in Lima, Peru. The analysis was confirmed by Polymerase Chain Reaction and automated sequencing of a liver biopsy sample, even though the serologic tests were negative. In conclusion, Bartonella spp. infection should have a particular diagnostic consideration in immunocompromised patients with fever of unknown origin and further investigation regarding the patient's past exposures with cats should also be elicited.

Acute lymphoblastic leukemia (ALL) is the most common childhood malignancy. Most ALL cases originate from immature B-cells (BCP-ALL) and are characterized by reoccurring structural genetic aberrations. These aberrations hold information of the pathogenesis of ALL and are used for risk stratification in treatment. Despite increased knowledge of genetic aberrations in pediatric T-cell ALL (T-ALL), no reliable molecular genetic markers exist for identifying patients with higher risk of relapse. The lack of molecular prognostic markers is also evident in patients with relapsed ALL. During the last decades, aberrant epigenetic mechanisms including DNA methylation have emerged as important components in cancer development. Telomere maintenance is another important factor in malignant transformation and is crucial for long-term cell survival. Like DNA methylation, telomere length maintenance has also been implicated to reflect outcomes for patients with leukemia. In this thesis, the prognostic relevance of DNA methylation and telomere length was investigated in pediatric ALL at diagnosis and relapse. The telomere length (TL) was significantly shorter in diagnostic ALL samples compared to normal bone marrow samples collected at cessation of therapy, reflecting the proliferation associated telomere length shortening. Prognostic relevance of TL was shown in low-risk BCP-ALL patients where longer telomeres at diagnosis were associated with higher risk of relapse. Genome-wide methylation characterization by arrays in diagnostic T-ALL samples identified two distinct methylation subgroups denoted CIMP+ (CpG Island Methylator Phenotype high) and CIMP- (low). CIMP- T-ALL patients had significantly worse outcome compared to CIMP+ cases. These results were confirmed in a Nordic cohort treated according to the current NOPHO-ALL2008 protocol.  By combining minimal residual disease (MRD) status at treatment day 29 and CIMP status at diagnosis we could further separate T-ALL patients into risk groups. Likewise, the CIMP profile could separate relapsed BCP-ALL patients into risk groups, where the CIMP- cases had a significantly worse outcome compared to CIMP+ cases.  From these data we conclude that DNA methylation subgrouping is a promising prognostic marker in T-ALL, as well as in relapsed BCP-ALL two groups where reliable prognostic markers are currently missing. By elucidating the biology behind the different CIMP profiles, the pathogenesis of ALL will be further understood and may contribute to new treatment strategies.

Orientador: Marcos Antônio de Oliveira / Resumo: L-asparaginases (ASNases) bacterianas são importantes biofármacos utilizados no tratamento de leucemia linfoide aguda (LLA), uma vez que este tipo tumoral é dependente da disponibilidade de asparagina (Asn) extracelular. As ASNases bacterianas são capazes de hidrolisar eficientemente Asn em ácido aspártico (Asp) e amônia (NH3), diminuindo a disponibilidade de Asn para células tumorais e induzindo apoptose. Comercialmente, indústrias farmacêuticas internacionais produzem ASNases de Escherichia coli e Erwinia chrysanthemi, entretanto, ASNase de nenhuma origem é produzida pelas indústrias farmacêuticas brasileiras. Adicionalmente, o tratamento com estas enzimas produz efeitos colaterais, entre eles imunogênicos, que estão relacionados com a alta massa molecular da enzima (140kDa) e neurológicos, atribuídos a atividade secundária de glutaminase (GLNase). Neste contexto, fontes alternativas destas enzimas e também a auto-suficiência em suas produções são importantes para mitigar os efeitos colaterais e evitar falhas no tratamento devido a flutuações internacionais de sua fabricação. Neste trabalho, realizamos a caracterização de uma ASNase de levedura, denominada de ASNaseM que compartilha elevada homologia (maior que 30% de identidade e 40% de similaridade) com as enzimas bacterianas utilizadas no tratamento da LLA e que possui todos os aminoácidos envolvidos na catálise conservados, sugerindo uma fonte alternativa potencial para o tratamento da LLA. Experimentos de cromatografia... (Resumo completo, clicar acesso eletrônico abaixo) / Bacterial L-asparaginases (ASNases) are important biopharmaceuticals used in the treatment of acute lymphoid leukemia (ALL), since this tumor type is dependent on the availability of extracellular asparagine (Asn). Bacterial ASNases are able to efficiently hydrolyze Asn in aspartic acid (Asp) and ammonia (NH3), decreasing the availability of Asn to tumor cells and inducing apoptosis. Commercially, international pharmaceutical industries produce ASNases from Escherichia coli and Erwinia chrysanthemi, however none ASNase is produced by the Brazilian pharmaceutical companies. Additionally, the treatment with these enzymes can produce side effects, among them immunogenic ones, that are related to the high molecular weight of the enzyme (140kDa) and neurological, attributed to the glutaminase secondary activity (GLNase), being glutamine (Gln) the most abundant amino acid in the bloodstream. In this context, alternative sources of these enzymes as also the self-sufficiency of the production are important to mitigate side effects and avoid treatment failures due to international fluctuations in their manufacture. In this work, we performed the characterization of a yeast ASNase, called ASNaseM, which shares high homology (higher than 30% identity and 40% similarity) with the bacterial enzymes used in the treatment of ALL, and which has all the amino acids involved in the catalysis conserved, suggesting a potential alternative source for the treatment of ALL. Molecular exclusion chro / Doutor

Acute lymphoblastic leukemia (ALL) is an aggressive hematologic malignancy that results from the unregulated growth of B-cell and T-cell lymphoid progenitors. Despite the implementation of risk-stratification and improved multi-agent therapeutic regimens, 20% of pediatric and 50% of adult patients fail to achieve remission and end up relapsing. NT5C2 (5’ cytosolic nucleotidase II) is the most frequently mutated gene specifically found in relapsed ALL. NT5C2 mutations are present in 20% of relapsed T-ALLs and 3-10% of relapsed B-ALLs and present as heterozygous gain of function alleles exhibiting increased nucleotidase activity. As NT5C2 can dephosphorylate and inactivate the cytotoxic metabolites generated by 6-mercaptopurine, a chemotherapy used in the treatment of ALL, these NT5C2 activating mutations can contribute to thiopurine chemotherapy resistance (Tzoneva, Perez-Garcia et al. 2013). Here we perform an extensive structure-function study to understand how relapse-associated NT5C2 mutations result in increased nucleotidase activity and contribute to chemotherapy resistance in ALL. Crystallization of 15 NT5C2 WT and mutant structures as well as enzymatic, structural modeling, and genetic screens identified three regulatory mechanisms of NT5C2, which are disrupted by these gain of function alleles. Class I NT5C2 mutations lock the protein in an active configuration through stabilization of the helixA region, which allows for substrate processing and catalysis. Class II NT5C2 mutations disrupt an intramolecular switch off domain involving the arm region and the intermonomeric positively charged pocket. And a single C-terminus truncating mutant creates a third class of mutations, which show increased nucleotidase activity due to the loss of the C-terminus blockade against allosteric activation. These studies provide new insight into the regulatory controls that mediate NT5C2 activity providing a framework for the development of targeted inhibitors for the treatment of relapsed ALL. In addition to looking at relapse associated NT5C2 mutations on a structural level, we also explored how NT5C2 mutations shape the clonal architecture and evolutionary dynamics during tumor initiation and disease progression in ALL. To formally address these questions, we developed a murine NOTCH1-driven T-ALL with conditional knock-in of the Nt5c2R367Q mutation, the most recurrent mutation found in relapsed ALL, from the endogenous locus. Using this model, we confirmed that Nt5c2+/R367Q lymphoblasts show increased resistance to 6-MP in vitro and in vivo. We also found that Nt5c2+/R367Q mutant lymphoblasts exhibit impaired cell fitness and decreased leukemia initiating cell capacity. Metabolomic profiling and guanosine rescue experiments show that this decrease in cell fitness is due to excess clearance of purine metabolites out of the cell as a result of deregulated Nt5c2 nucleotidase activity. However, in the context of 6-MP therapy, Nt5c2+/R367Q mutant cells are positively selected for in mixed population studies in vitro and in vivo. These results identify a clear selective advantage for NT5C2 mutant cells in the context of 6-MP chemotherapy. In addition, NT5C2 mutant chemoresistant cells show collateral sensitivity to inhibition of inosine monophosphate dehydrogenase (IMPDH) with mizoribine, which further disrupts guanosine production pointing to a potentially selective therapy against NT5C2 mutant cells. We also show here the initial development of a small molecule NT5C2 inhibitor for the treatment of relapsed ALL. Using a malachite green based NT5C2 nucleotidase assay, we performed a small molecule high throughput assay and identified HTP_2 as a lead compound with low micromolar inhibitory activity against NT5C2 R367Q mutant recombinant protein. HTP_2 can reverse 6-MP resistance in Nt5c2+/R367Q mouse lymphoblasts and NT5C2 R29Q mutant expressing human cell lines. Interestingly, HTP_2 treatment also results in increased sensitivity to 6-MP therapy in NT5C2 wild-type cells, suggesting a role for wild-type NT5C2 activity in the clearance of 6-MP and supporting a potential therapeutic use for NT5C2 inhibitors in potentiating the effects of 6-MP based chemotherapy in NT5C2 wild-type cells as well. NT5C2 knockdown cells and Nt5c2 knockout mice show no apparent toxicities suggesting that systemic inhibition of NT5C2 could be fairly well tolerated. In all, this work presents a framework for the development of a high affinity NT5C2 inhibitor for the reversal of 6-MP resistance in relapsed ALL patients. These studies presented here address the role of NT5C2 mutant proteins as drivers of resistance and as therapeutic targets in relapsed ALL. Improved understanding of the molecular mechanisms responsible for increased NT5C2 nucleotidase activity and on the process of clonal evolution during disease progression provide important insight into the mechanism driving ALL resistance and relapse. The identification of IMPDH inhibition as a collateral vulnerability in NT5C2 mutant ALL cells and the development of a first-in-class NT5C2 inhibitor serve as framework for the development of new combination therapies aimed at curtailing the emergence of these thiopurine-resistant relapse driving clones in ALL.

by Betty Shuc Han Wills. / Year shown on spine: 1997. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 117-128). / ACKNOWLEDGMENT --- p.i / ABSTRACT --- p.ii / TABLE OF CONTENTS --- p.iv / LIST OF APPENDICES --- p.viii / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter CHAPTER 2 --- LITERATURE REVIEW --- p.6 / Parental Responses to the Diagnosis of Acute Lymphocytic Leukaemia (ALL) --- p.7 / Disclosure Of the Child's Diagnosis --- p.10 / Impact of Cancer Treatment on Parents --- p.14 / Sources of Support for Parents --- p.18 / Coping Strategies of Parents of Children With ALL --- p.20 / Coping With The Uncertainty of the Disease --- p.23 / Research Studies Involving Chinese Parents --- p.24 / Summary Of Issues From Literature Review --- p.27 / Chapter CHAPTER 3 --- METHODOLOGY / Research Design --- p.29 / Sampling --- p.31 / Data Collection Method --- p.32 / Data Collection Procedure --- p.34 / Ethical Considerations --- p.38 / Pilot Study --- p.40 / Data Analysis --- p.42 / Issues of Reliability and Validity --- p.45 / Validity --- p.45 / Reliability --- p.48 / Chapter CHAPTER 4 --- RESULTS & DISCUSSION / Introduction --- p.50 / Chapter (I). --- Parents' Profile --- p.51 / Demographic Characteristics Of The Parents / Chapter (II). --- Major categories Corresponding To Interviewing The Mothers --- p.54 / Initial Reactions of the Child's Confirmed Diagnosis --- p.55 / Unpreparedness for the child's Diagnosis / Suddenness of the Diagnosis --- p.56 / Physical and psychological reactions to the child's Diagnosis --- p.58 / Sources of Support for the Mothers --- p.62 / The mothers' main source of support / Other sources of support for the mothers --- p.64 / Disclosure Of Child's Diagnosis --- p.66 / Disclosure of the child's diagnosis to the child / Disclosure of the child's diagnosis to members of the immediate and extended families --- p.68 / Disclosure of child's diagnosis to non-family members --- p.70 / Uncertainty Brought On By The Illness --- p.71 / Waiting for confirmation of diagnosis / Uncertainty about the success of treatment --- p.73 / Uncertainty about the child's future --- p.74 / Changes In The Family Routine --- p.75 / Needed to normalise family life / Chapter (III). --- Major Categories Corresponding to Interviewing The Fathers Initial reactions to the child's confirmed diagnosis of ALL --- p.78 / Suddenness of diagnosis --- p.79 / Physical and psychological reactions to the diagnosis --- p.80 / Disclosure Of The Child's Diagnosis --- p.82 / Disclosure of the child's diagnosis to the child / Disclosure of the child's diagnosis to members of the immediate and extended family --- p.85 / Disclosure of the child's diagnosis to non-family members --- p.86 / Sources Of Support For The Fathers --- p.87 / Support from immediate and extended families / Support from medical professionals --- p.89 / Support from friends --- p.90 / Changes In The Family Routine --- p.91 / Coping Strategies Utilised By The Fathers --- p.92 / Open communication / Use of religious beliefs and rituals --- p.93 / Chapter (IV). --- Comparison Of Categories Found Between The Mothers And The Fathers --- p.95 / Initial reactions to the child's confirmed diagnosis of ALL / Disclosure of the child's diagnosis --- p.96 / Sources of support for the parents --- p.100 / Changes in the family routine --- p.101 / Summary of findings --- p.103 / Chapter (V). --- Differences between the initial and second interviews --- p.105 / Chapter CHAPTER FIVE --- CONCLUSION / Limitations Of The Study --- p.108 / Implications For Nursing Practice --- p.112 / Recommendations For Future Research --- p.114 / Conclusion --- p.115 / REFERENCES --- p.117 / APPENDIX I - PERSONAL DATA FORM --- p.129 / APPENDIX II - INTERVIEW SCHEDULE --- p.130 / APPENDIX III - CONSENT FORM --- p.134 / APPENDIX IV - SAMPLE SCRIPT OF INTERVIEWS WITH MOTHERS --- p.135 / APPENDIX V - MATRICES ON MOTHERS AND FATHERS --- p.140 / APPENDIX VI - SAMPLE OF FIELD NOTES FOR MOTHERS AND FATHERS --- p.143

T-cell acute lymphoblastic leukemia (T-ALL) is a lymphoid disorder that results from an over proliferation of immature lymphocytes in the blood and bone marrow. It has been determined that 60% of patients stricken with T-ALL aberrantly express TAL-1 and have been shown to respond poorly to chemotherapy. This research sought to determine if TAL-1 influences the expression of the Bcl-2 family members Bcl-2 (anti-apoptotic), Bad and Bax (pro-apoptotic). TAL-1 and Bcl-2 levels were elevated while Bad and Bax levels were lower in etoposide-treated Jurkat cells as compared to TRAIL-treated and dual-treated Jurkat cells in which TAL-1 and Bcl-2 levels were lower while Bad and Bax levels were elevated. These results suggest TAL-1 up-regulates Bcl-2 and suppress Bad and Bax expression in response to etoposide treatment, thus inducing an anti-apoptotic response in the cell. These results also suggest that TRAIL and the dual treatment of etoposide and TRAIL down-regulates TAL-1 and Bcl-2 expression while up-regulating Bad and Bax, thus inducing a pro-apoptotic response in the cell. / Department of Biology

Tal-1 is a transcription factor that is frequently ectopically expressed in the majority of cases of T-cell acute lymphoblastic leukemia (T-ALL). The ectopic expression of Tal-1 in patients with ALL has been found to decrease susceptibility to chemotherapeutic drugs and apoptosis. Thus, this study focuses on the effects of endogenously expressed Tal-1 in the Jurkat cell line on three Bcl-2 family members (Bcl-2, Bcl-xL, and Bid) and the inhibition of apoptosis and cell viability when exposed to apoptosis inducing drugs such as etoposide. The data obtained indicate that when treated with etoposide for 12 h Jurkat cells endogenously expressing Tal-1 have an 81% higher level of anti-apoptotic Bcl-2 expression, an 18% lower level of anti-apoptotic Bcl-xL, expression, and a 31% lower level of pro-apoptotic Bid expression compared to Jurkat cells lacking Tal-1 expression. The data also demonstrates that Jurkat cells endogenously expressing Tal-1 have a 15.94% lower amount of cell death after treatment with etoposide for 12 h and a 20.34% lower amount of cell death after treatment with etoposide for 24 h when compared to Jurkat cells that lack Tal-1 expression. Thus, the endogenous expression of Tal-1 increases the amount of the anti-apoptotic Bcl-2 expression and decreases the amount of the pro-apoptotic Bid creating an overall anti-apoptotic signal within the cell. / Department of Biology

Childhood acute lymphoblastic leukemia (ALL) is the most common malignancy in children. Several proto-oncogenes that encode nuclear proteins are activated by various chromosomal translocations in ALL including TALI, TAL2, and LMO1 and LMO2. Ectopic TALI expression is observed in about 50 % of T-ALL and is the most common genetic anomaly associated with this pathology. Of interest to the present work is the characterization of various multiprotein complexes and protein protein interactions that drive T-ALL progression (as it relates to TALI and LMO1) and over expression of TALI and LMO1 has been shown to have inhibitory effects on apoptosis. Recent data suggests possible interactions between these two oncoproteins and the protein product of the lipoprotein lipase (LPL) gene. Lipoprotein lipase has a complex pattern of regulation and can be regulated in different ways including down-regulation upon induction of TNF-a in 3T3-L1 cells. Thus, this study was undertaken to determine if LPL is expressed in cells over expressing TAL1 and LMO1. Results from this study demonstrated an increase in LPL expression at both transcriptional and translational level in cells engineered to express TAL1 alone and TAL1 and LMO1 together. This finding is a step forward to understanding mechanisms that result in apoptosis prevention in T-ALL. Therefore, the apoptosis preventive role seen in cells that over express TAL and LMO1 and the presence of LPL in the same cell line, theorizes an apoptosis preventive role for lipoprotein lipase as well. / Department of Physiology and Health Science

Currently, the intensive chemotherapy remains the first line treatment for B cell acute lymphoblastic leukemia (B-ALL). Although these regimens have significantly improved patient outcomes, their use is associated with debilitating morbidities and fatal relapses, highlighting the great need in new agents that target essential survival signals in leukemia. Thus, the overall goal of my project was to gain insights into the signaling abnormalities that regulate aberrant proliferation and survival of B-ALL cells in an effort to identify novel targets in this malignancy. This study demonstrated that pre-B cell receptor (pre-BCR)-independent spleen tyrosine kinase (SYK) activity was required for the survival and proliferation of a p53-/-PrkdcSCID/SCID mouse model of B-ALL. I extended this discovery to human disease, demonstrating that SYK was activated in primary B-ALL, independent of the pre-BCR expression. The small molecule SYK inhibitor fostamatinib (fosta) significantly attenuated proliferation of 79 primary diagnostic B-ALL samples at clinically achievable concentrations. Importantly, fosta treatment reduced dissemination of engrafting B-ALL cells into the spleen, liver, kidney and central nervous system (CNS) in a NOD.Prkdcscid/scidIl2rgtm1Wjl/SzJ xenotransplant model of B-ALL. Analysis of signaling abnormalities using a high-throughput phospho-flow cytometry platform demonstrated that pediatric and adult B-ALL samples exhibit variable basal activation of BCR, iii PI3K/AKT/mTOR, MAPK and JAK/STAT pathways. Importantly, we identified that fosta-mediated inhibition of SYK, PLC2, CRKL and EIF4E phosphorylation in B-ALL was predictive of its anti-leukemic activity, and was distinct from the cellular actions of other small molecule inhibitors of key nodal signaling pathways. Examination of molecular mechanism of fosta action by gene expression profiling revealed transcriptional effects of fosta treatment that included, most notably, potent inhibition of pathways involved in lymphocyte activation and inflammation. In conclusion, this study demonstrates that SYK signaling is crucial for B-ALL survival and provides detailed characterization of cellular and molecular mechanisms of fosta action in B-ALL. These data argue in favor of testing small molecule SYK inhibitors in pediatric and adult B-ALL.